Adjuvancy and immune potentiating properties of natural products of Onchocerca volvulus

ABSTRACT

The present invention relates to a method for potentiating a specific immune response to an antigen in a mammal in need thereof. The method comprises administering to the mammal an effective amount of Ov-ASP, or at least one subunit of Ov-ASP, and an antigenic moiety.

This application claims benefit of U.S. Provisional Application No.60/580,254, filed Jun. 15, 2004, incorporated herein by reference.

BACKGROUND OF THE INVENTION

The increased threat of a bioterrorist attack in recent years highlightsthe critical need for the development of potent vaccine formulations toprotect the susceptible population. Vaccine formulations containantigens that induce immunity against pathogenic agents. However, immuneresponses to many antigens, while detectable, are frequently ofinsufficient magnitude to afford protection against a disease processmediated by the agents expressing those antigens. In such situations, itis necessary to include an adjuvant along with the antigen in thevaccine formulation.

An adjuvant is a compound that, when used in combination with specificvaccine antigens, potentiates the resultant immune response. Themechanism of action of adjuvants is not precisely known, and may not bethe same for all adjuvants. However, it is believed that adjuvantsprolong the bioavailability of an antigen. Adjuvants also seem toincrease the size of the antigen, thus increasing the likelihood ofphagocytosis. Additionally, most adjuvants have a stimulatory effect onthe cell-mediated branch of the immune system, i.e., on T lymphocytes (Tcells).

There are two well-defined subpopulations of T cells: T cytotoxic (Tc)cells and T helper (Th) cells. T cytotoxic cells kill intracellularpathogens. On the other hand, Th cells exert most of their functionsthrough secreted cytokines. T helper cells are further divided into Th1and Th2 cell types. Differences in cytokine-secretion patterns of the Thcell types determine the type of immune response made to a particularantigen challenge.

In general, Th1 cells stimulate cytotoxic responses againstintracellular viruses, bacteria and protozoa via secretion ofinterferon-gamma (IFN-γ) and other pro-inflammatory cytokines. Thecytotoxic responses include the activation of Tc cells. In contrast, Th2cells are induced by allergens and helminth parasites, and arecharacterized by the secretion of interleukins, e.g., IL-4, IL-5, etc.Both Th cell types stimulate the humoral branch of the immune system,i.e., the B lymphocytes.

Different pathogens elicit different types of cell-mediated immuneresponses. For example, infecting mice with a helminth parasitepolarizes the immune response to Th2 activation. In some cases, thepolarization is so potent that a Th1-dominant response to an infectiouspathogen can be inhibited by the introduction of a helminth parasite.(Brady et al “Fasciola hepatica suppresses a protective Th1 responseagainst Bordetella pertussis” Infect. Immun. 67: 5372-5378 (1999).)Similarly, a Th1-mediated mouse autoimmune disease can be ablated byintroducing a helminth parasite into mice (Cooke et al. “Infection withSchistosoma mansoni prevents insulin dependent diabetes mellitus innon-obese diabetic mice” Parasite Immunol. 21:169-176 (1999)).

Additionally, the anti-inflammatory properties of the products of twohelninth parasites have been shown to be capable of down-modulatinginflammatory Th1responses in mice. In particular, body fluid from thepig roundworm parasite, Ascaris suum, potently stimulates cytokinescharacteristic of Th2 cells. (Paterson et al., “Modulation of aHeterologous Immune Response by the Products of Ascaris suum” Infect.Immuunol. 70:6058-67 (2002)). Also, a secreted glycoprotein product,ES-62, of a rodent parasite has been found to have broadanti-inflammatory properties that inhibit Th1 cytokine production inexperimentally-induced arthritis in mice (McInnes et al., “A NovelTherapeutic Approach Targeting Articular Inflammation Using the FilarialNematode-Derived Phosphorylcholine-Containing Glycoprotein ES-62” J.Immuunol. 171:2127-33 (2003)). This product is currently being developedas a novel anti-inflammatory therapeutic.

Recently, two helminth products have been reported as acting asadjuvants. Both are strong inducers of Th2 responses to bystanderproteins in a vaccine. In particular, proteins secreted by adultNippostrongylus brasiliensis (a parasite of rodents) were found to bestrong inducers of Th2 responses in mice immunized with an unrelatedprotein (Holland et al., “Proteins secreted by the parasitic nematodeNippostrongylus brasiliensis act as adjuvants for Th2 respones” Eur. J.Immunol. 30 (7):1977-1987 (2000)). Similarly, lacto-N-fucopentaose III,a carbohydrate found on the surface of the eggs of a human parasite,Schistosoma mansoni, acted as a Th2 adjuvant for a bystander proteinwhen injected into mice (Okano et al., “Lacto-N-fucopentaose III Foundon Schistosoma mansoni Egg Antigens Functions as Adjuvant for Proteinsby Inducing Th2-Type Response” J. Immunol. 167:442-450 (2001)).

Until the present invention, products from helminths have been found tobe potently Th2 dominant. Accordingly, their use as adjuvants has beento induce the Th2 cell type responses. Although Th2 cell type activationis important, Th1 cell type activation is critical for the efficacy ofcertain vaccines. In addition to providing a different cytokine profilethan that provided by Th2 cells, Th1 cells activate cytotoxic effectormechanisms which Th2 cells do not activate.

Moreover, other adjuvants presently used in human vaccines also are noteffective in stimulating cytotoxic responses to intracellular pathogens.These adjuvants include aluminum salts, e.g., aluminum potassiumsulfate, aluminum phosphate and aluminum hydroxide. Without the abilityto stimulate cytotoxic responses to intracellular pathogens, the use ofsuch adjuvants is limited.

In additional to protecting against infectious diseases, vaccination isbecoming significant in other developing technologies. Thesetechnologies include, for example, vaccination against syngeneic tumors.In such new approaches, it is important to be able to induce differenttypes of immune responses.

Accordingly, there is a critical need for safe and effective adjuvantsand therapeutics capable of boosting immune responses to a wide varietyof pathogens and against tumors. There is a particular need foradjuvants that boost Th1 cell type responses.

SUMMARY OF THE INVENTION

In one embodiment, the present invention relates to a vaccinecomposition or immunogenic composition which comprises an antigenicmoiety; and an adjuvant comprising an effective amount of Ov-ASP, or ofat least one subunit of Ov-ASP. Ov-ASP includes Ov-ASP-1, Ov-ASP-2 andOv-ASP-3.

In another embodiment, the present invention relates to a method forpotentiating a specific immune response to an antigen in a mammal inneed thereof. The method comprises administering to the mammal aneffective amount of Ov-ASP, or at least one subunit of Ov-ASP; and anantigenic moiety.

In a further embodiment, the present invention relates to a method forstimulating a cellular response with cytokine secretion in a mammal inneed thereof. The method comprises administering to the mammal aneffective amount of Ov-ASP, or at least one subunit of these proteins,wherein the cytokine secretion is stimulated.

In an additional embodiment, the present invention relates to a methodof generating an immune response or vaccinating a mammal in need thereofagainst onchocerciasis. The method comprises administering to the mammalan effective amount of Ov-ASP, or antigenic fragments of Ov-ASP, and apharmaceutically-acceptable carrier.

In another aspect, the present invention relates to a method ofpreventing SARS in a mammal in need thereof. The method comprisesadministering to the mammal a vaccine composition comprising a SARS-CoVpolyamino acid, and an effective amount of Ov-ASP, or at least onesubunit of Ov-ASP. In another aspect, the present invention relates to amethod of preventing HIV in a mammal in need thereof. The methodcomprises administering to the mammal a vaccine composition comprisingan HIV-1 polyamino acid, and an effective amount of OV-ASP, or at leastone subunit of Ov-ASP.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1. Cytokine secretion induced by rOv-ASP-1 (5 μg/mL) from PBMCobtained from individuals (n=14) never exposed to Onchocerca volvulus.Cells were incubated with rOv-ASP-1 (+) or culture medium alone (−).*=P<0.05 versus cells in culture medium alone. Values are the mean±SD.

FIG. 2. Inhibition of LPS activity using polymyxin B (5 and 20 μg/mL)has no effect on the bioactivity of rOv-ASP-1 (5 μg/mL) on human PBMC (3donors). rOv-ASP-1 was pre-incubated with polymyxin B for 1 hour at roomtemperature prior to adding to PBMC. Values are the mean±SD.

FIG. 3. Cytokines produced by spleen cells from mice immunized with PBSor rOv-ASP-1 without adjuvants and stimulated in vitro with 5 μg/mLrOv-ASP-1. Values are obtained from spleen cells pooled within eachtreatment group and represent the mean of triplicate cultures.

FIG. 4. Mean anti-OVA IgG1 and IgG2a in mice (n=5/group) immunized withcontrol treatments (PBS, OVA, alum, MPL+TDM, rOv-ASP-1) or OVA combinedwith alum or MPL+TDM or the test adjuvant, rOv-ASP-1. Antibody amountsare expressed as optical density (OD) in the ELISA assay.

FIG. 5. Mean anti-OVA IgG1 and IgG2a titers in mice (n=5/group) bledpre-immunization (Pre) or after immunization with control treatments(PBS, OVA) or OVA combined with the test adjuvant, rOv-ASP-1 (25μg/mouse), which was either treated (LPS−) or untreated (LPS+) withLPS-removing gel. The same symbols apply in both graphs. The end pointanti-OVA IgG1 titer was 512,000 and the end point anti-OVA IgG2a titerwas 128,000.

FIG. 6. Cytokines produced by spleen cells from mice immunized with OVAwith or without adjuvants or relevant control treatments andre-stimulated in vitro with 5 μg/mL OVA. Values are obtained from spleencells pooled within each treatment group and represent the mean oftriplicate cultures.

FIG. 7. Mean amounts of anti-SC-1 total IgG in mouse sera (n=5/group)after immunization with control treatments (antigens or adjuvants alone)or antigens formulated with MPL+TDM or the test rOv-ASP-1 adjuvant.Antibody amounts are expressed as optical densities (OD) in the ELISAassays. The reciprocal dilutions of serum are indicated on the x axis. Tend points are denoted; 250,000 in the presence of rOv-ASP-1 and 64,000in the presence of MPL+TDM.

FIG. 8. Mean amounts of anti-FLSC total IgG in mouse sera (n=5/group)after immunization with control treatments (antigens or adjuvants alone)or antigens formulated with MPL+TDM or the test rOv-ASP-1 adjuvant.Antibody amounts are expressed as optical densities (OD) in the ELISAassays. The reciprocal dilutions of serum are indicated on the x axis. Tend points are denoted; 1,024,000 in the presence of rOv-ASP-1 and1,024,000 in the presence of MPL+TDM.

DETAILED DESCRIPTION OF THE INVENTION

The present invention comprises pharmaceutical compositions and methodsto stimulate, i.e., induce and/or potentiate, immune responses inmammals. The invention includes the unexpected discovery that proteinsfrom a helminth parasite, Onchocerca volvulus, can stimulate variousaspects of the mammalian immune response.

The proteins used in the pharmaceutical compositions and methods of theinvention are members of the Ov-ASP (Onchocerca volvulusactivation-associated secreted protein) family. Native Ov-ASPs arelocated in secretory granules of the glandular esophagus and the surfaceof the infective third stage larvae of the helminth Onchocerca volvulus.

Members of the Ov-ASP family include Ov-ASP-1, Ov-ASP-2 and Ov-ASP-3.The sequence of Ov-ASP-1 is shown in SEQ ID NO: 1. The sequence ofOv-ASP-2 is shown in SEQ ID NO:2. The sequence of Ov-ASP-3 is shown inSEQ ID NO:3. Ov-ASP used in the compositions and methods of theinvention need not be 100% identical to SEQ ID NO:1, SEQ ID NO:2 or SEQID NO:3, as long as the protein retains the immune-stimulatingproperties of SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3. For example,Ov-ASP, for the purposes of this specification, is approximately 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ IDNO:1, SEQ ID NO:2 or SEQ ID NO:3.

One or more subunits (i.e., fragments) of Ov-ASP can be used in thecompositions and methods of this invention. A subunit can be any lengthwhich produces the desired stimulation of an immune response (i.e., anactive subunit). The minimum number of amino acids of a subunitincludes, for example, at least about twenty, thirty, forty, fifty,sixty, seventy, eighty, and ninety amino acids. The maximum number ofamino acids of a subunit includes, for example, at most about twohundred fifty three, two hundred fifty, two hundred forty, two hundredthirty, two hundred twenty, two hundred ten, two hundred, one hundredninety, one hundred eighty, one hundred seventy, one hundred sixty, onehundred fifty, one hundred forty, one hundred thirty, one hundredtwenty, one hundred ten, and one hundred amino acids. A suitable rangeof amino acids includes any number from the minimum and any number fromthe maximum.

For the purposes of this specification, “Ov-ASP” includes a full lengthOv-ASP-1, or one or more subunits of a full length Ov-ASP-1; a fulllength Ov-ASP-2, or one or more subunits of a full length Ov-ASP-2; or afull length Ov-ASP-3, or one or more subunits of a full length Ov-ASP-3.

Ov-ASP, and subunits of these proteins, can be prepared by methods knownin the art. Preferably, the proteins are produced recombinantly. Forexample, recombinant protein, rOv-ASP-1, was expressed in E. coli usingcDNA encoding Ov-ASP-1 (Ov-ASP-1: GenBank accession number AF020586).This recombinant protein has a molecular weight of 24,871 Da.Recombinant protein, rOv-ASP-2, was expressed in E. coli using cDNAencoding Ov-ASP-2 (Ov-ASP-2: GenBank accession number H39490). Thisrecombinant protein has a molecular weight of 29,047 Da. Recombinantprotein, rOv-ASP-3, was expressed in E. coli using cDNA encodingOv-ASP-3 (Ov-ASP-3: GenBank accession number AA917267). This recombinantprotein has a molecular weight of 24,744 Da. See Tawe et al.,“Angiogenic activity of Onchocerca volvulus recombinant proteins similarto vespid venom antigen 5” Mol. Biochem. Parasitol. 109: 91-99 (2000).The sequences and methods of providing Ov-ASP from U.S. Pat. No.6,723,322 (Lustigman et al.) are incorporated herein by reference.

Ov-ASP can also be obtained by isolating the protein directly fromOnchocerca volvulus by standard methods. Some suitable methods includeprecipitation and liquid chromatographic protocols, such as ionexchange, hydrophobic interaction and gel filtration. (Methods Enzymol.182 (Guide to Protein Chemistry, Deutscher, Ed. Sec. VII) 309 (1990);and Scopes, Protein Purification. Springer-Verlag, N.Y. (1987).) Ov-ASPcan also be obtained by separating the protein on preparative SDS-PAGEgels, slicing out the band of interest and electroeluting the proteinfrom the polyacrylamide matrix.

Ov-ASP can also be obtained by synthesizing the protein from individualamino acid residues, as known in the art. (Stuart and Young “Solid PhasePeptide Synthesis,” 2^(nd) Ed., Pierce Chemical Co. (1984).)

The administration of Ov-ASP in the methods of the invention can beeffected by administering the protein itself, or by introducing anucleic acid molecule encoding the protein in a manner permittingexpression of the protein. Preferably, the nucleic acid molecule is inthe form of a recombinant expression vector, such as, for example, apurified plasmid. After administration of the expression vector into amammalian cell, Ov-ASP is expressed intracellularly.

Recombinant vectors can also contain a nucleotide sequence encodingsuitable regulatory elements so as to effect expression of the vectorconstruct in a suitable host cell. Those skilled in the art willappreciate that a variety of enhancers and promoters are suitable foruse in the constructs of the invention, and that the constructs willcontain the necessary start, termination, and control sequences forproper transcription and processing of the nucleic acid sequenceencoding an Ov-ASP when the recombinant vector construct is introducedinto a subject.

Vaccine Compositions or Immunogenic Compositions Comprising Ov-ASP as anAdjuvant

In one embodiment, the invention relates to vaccine compositions orimmunogenic compositions comprising Ov-ASP and an antigenic moiety.Ov-ASP is used as an adjuvant in these compositions. As an adjuvant,Ov-ASP potentiates an immune response to antigens which are unrelated toOv-ASP.

In this embodiment, vaccine compositions or immunogenic compositionswhich comprise at least one antigenic moiety and an effective amount ofOv-ASP are provided. The vaccines of the invention can be prophylacticvaccines or therapeutic vaccines. A prophylactic vaccine prevents adisease from occurring by priming the immune system to respond to anantigen. A therapeutic vaccine is given after infection to reduce orarrest disease progression by producing or reinforcing an immuneresponse.

The ratio by weight of the antigenic moiety to Ov-ASP in the vaccinecompositions or immunogenic compositions can be any ratio which allowsfor the potentiation of a specific immune response. The amount of Ov-ASPto be added to a particular antigenic moiety depends on several factors,as would be known by a skilled artisan. Factors include, for example,the age and weight of the subject mammal, the mode of administration ofthe composition, the inherent immunogenicity of the particular antigen,the desired form of the response (elevation of titer, prolongation ofthe response, or both), the presence of carriers, and otherconsiderations that will be apparent to those skilled in the art. Theamount can be determined by routine experimentation. For example, theratio by weight of an antigenic moiety to Ov-ASP can range from about4:1 to about 1:1, or from about 4:1 to about 1:4.

The antigenic moiety of the present invention can be an antigen or anucleic acid molecule that encodes an antigen. An antigen is a substanceto which a specific immune response in a mammal can be induced. That is,an antigen is immunogenic. A specific immune response includes a humoraland/or a cell-mediated immune response directed specifically against theantigen. For the purposes of this specification, an antigen includessubstances that are capable of eliciting immune responses whenadministered to a mammal by itself, and substances that are capable ofeliciting immune responses only when administered to a mammal togetherwith Ov-ASP.

Antigens can, for example, be immunogenic polyamino acids. Polyaminoacids include oligopeptides, polypeptides, peptides, proteins andglycoproteins. The polyamino acid can be a naturally-occurring isolatedproduct, a synthetic product, or a genetically engineered polyaminoacid.

The length of a polyamino acid is not critical as long as the polyaminoacid is immunogenic when administered along with Ov-ASP. Therefore, thepolyamino acid contains a sufficient number of amino acid residues todefine at least one epitope of an antigen. Methods for isolating andidentifying immunogenic fragments from known immunogenic proteins aredescribed by Salfeld et al. in J. Virol. 63:798-808 (1989) and by Isolaet al. in J. Virol. 63:2325-2334 (1989).

If a polyamino acid defines an epitope, but is too short to beimmunogenic, it can be conjugated to a carrier molecule. Some suitablecarrier molecules include keyhole limpet hemocyanin, Ig sequences, TrpE,and human or bovine serum albumin. Conjugation can be carried out bymethods known in the art. One such method is to combine a cysteineresidue of the fragment with a cysteine residue on the carrier molecule.

Antigens can also be a lipid, a lipopolysaccharide (glycolipid) or apolysaccharide. The length of these compounds is not critical as long asthe compound induces an immune response. These compounds can also bechemically linked to protein carrier molecules in order to enhanceimmunogenicity. For example, a polysaccharide antigen, such as abacterial capsular polysaccharide or fragment thereof, can be linked toa protein carrier molecule to form a glycoconjugate. Methods forpreparing conjugates of bacterial capsular polysaccharide and proteincarrier molecules are well known in the art, and can be found, forexample, in Dick and Burret, Contrib Microbiol Immunol. 10:48-114 (CruseJ M, Lewis R E Jr., eds; Basel Kruger (1989)).

Antigens can be derived from various sources. Antigens are commerciallyavailable or can be produced as known by skilled artisans.

For example, antigens can be produced or derived from pathogenicmicroorganisms. Examples of microorganisms include viruses, e.g.,polyoma viruses; bacteria; mycoplasmas; fungi; protozoa; and otherinfectious agents. An antigen can be a whole microorganism. For example,an antigen can be a modified-live (i.e., attenuated) microorganism or akilled microorganism. An antigen can also be an immunogenic component ofa microorganism, or a product of a microorganism. For example, theantigen can be all or part of a protein, glycoprotein, glycolipid,polysaccharide or lipopolysaccharide which is associated with themicroorganism.

Pathogenic microorganisms from which antigens can be produced or derivedfor vaccine purposes are well known in the field of infectious diseases.Suitable pathogenic microorganisms are listed in, for example, MedicalMicrobiology, Second Edition, (1990) J. C. Sherris (ed.), ElsevierScience Publishing Co., Inc., New York, and Zinsser Microbiology, 20thEdition (1992), W. K. Joklik et al. (eds.), Appleton & Lange PublishingDivision of Prentice Hall, Englewood Cliffs, N.J.

Examples of microorganisms of particular interest for human vaccinesinclude human immunodeficiency virus (HIV), coronaviruses which causesevere acute respiratory syndrome (SARS), Chlamydia, Haemophilusinfluenzae, Helicobacter pylori, Moraxella catarrhalis, Neisseriagonorrhoeae, Neisseria meningitidis, Salmonella typhi, Streptococcuspneumoniae, herpes simplex virus, a rhabdovirus, human papilloma virus,influenza, measles, respiratory syncytial virus, rotavirus, Norwalkvirus, hepatitis A virus, hepatitis B virus, hepatitis C virus,tuberculosis-causing Mycobacterium, polio virus and smallpox virus.

An example of one of the preferred antigens that can be used in thepharmaceutical compositions of the present invention is a SARS-CoVpolyamino acid. An example of a SARS-CoV polyamino acid is the SARS-CoVSC-1 peptide (also known as CP-1, GenBank accession number: AY274119).Another example of one of the preferred antigens is an HIV-1-CD4polyamino acid. An example of a HIV-1-CD4 polyamino acid is theHIV-1-CD4 FLSC polypeptide. (Fouts et al, “Expression andcharacterization of a single-chain polypeptide analogue of the humanimmunodeficiency virus type 1 gp120-CD4 receptor complex” J. Virol. 74:11427-11436 (2000).) An example of another preferred antigen is derivedfrom E6 and E7 proteins of human papilloma virus (HPV), in particularfrom HPV-16.

Antigens for use in the present invention can also be derived fromallergens. Most allergens are small proteins or protein-bound substancesthat are capable of producing hypersensitivity. Examples of allergensinclude animal dander; plants, e.g., rye grass, ragweed, timothy grass,birch trees, etc.; insect products, e.g., venom, dust mites, etc.; food;egg albumin; and various other environmental sources.

Antigens also include polyamino acids native to the mammal beingtreated. Such self-polyamino acids include, for example, antigensassociated with tumors. Examples of such antigens include proteinsderived from growth factors, growth factor receptors, andoncogene-encoded proteins. Examples of growth factor receptors includeEGF receptors (HER1, HER2, HER3 and HER4), including the Neu proteinassociated with breast tumors, and transferrin growth factor, i.e., p97.Examples of oncogene-encoded proteins include oncofetal tumor antigens,e.g., alpha-fetoprotein and carcinoembryonic antigen.Melanoma-associated oncofetal antigens include MACE-1, MAGE-3, BAGE,GAGE-1, and GAGE-2.

Additionally, whole lysed tumor cells can be used thereby producingvaccines which comprise a collection of antigens. Examples include lysedcells from human melanoma cell lines and from human prostrate celllines. Whole cells can be derived from the mammalian subject beingtreated, or can be derived from another subject.

For the purposes of this specification, the antigenic moiety alsoincludes nucleic acid molecules which encode an antigen. The nucleicacid molecule is preferably in the form of a recombinant expressionvector, such as, for example, a purified plasmid. After administrationof the expression vector into a mammalian cell, the antigen is expressedintracellularly.

Recombinant vectors can also contain a nucleotide sequence encodingsuitable regulatory elements so as to effect expression of the vectorconstruct in a suitable host cell. Those skilled in the art willappreciate that a variety of enhancers and promoters are suitable foruse in the constructs of the invention, and that the constructs willcontain the necessary start, termination, and control sequences forproper transcription and processing of the nucleic acid sequenceencoding an antigen when the recombinant vector construct is introducedinto a subject.

The antigenic moiety and Ov-ASP can both be in protein form, or they canboth be in nucleic acid form. If the antigenic moiety and Ov-ASP areboth nucleic acids, they can both be on the same vector, or differentvectors. Alternatively, the antigenic moiety can be a protein antigenand Ov-ASP can be in nucleic acid form; or the antigenic moiety can bein nucleic acid form and Ov-ASP can be a protein.

Methods of Potentiating Specific Immune Responses

The present invention includes methods of potentiating a specific immuneresponse to an antigen in a mammal in need thereof. The methods compriseadministering to the mammal an effective amount of Ov-ASP, or at leastone subunit thereof; and an antigenic moiety. Ov-ASP, its subunits, andantigenic moieties suitable for use in the methods of the invention havebeen described above.

Ov-ASP, or its subunits; and the antigenic moieties can beco-administered separately, or as a vaccine composition or immunogeniccomposition, e.g., as described above.

Specific immune responses include humoral and cell-mediated responses.Humoral responses are mediated by B lymphocytes. Cell-mediated responsesinclude the activation of T cells, including Th1, Th2 and Tc cells.

Ov-ASP can potentiate both humoral and cell-mediated responses,including Th1 and Th2 responses. Ov-ASP is particularly effective inpotentiating Th1 responses, which in turn potentiate Tc responses. Thepotentiation of Th1 responses are particularly effective for tumorassociated antigens since most of such antigens are able to induce onlylow frequency, low-avidity transient T-cell responses, which are biasedtoward Th2-type cells.

An effective amount of Ov-ASP is an amount that potentiates a specificimmune response in a mammal. A potentiation of a specific immuneresponse is an increase in the magnitude of the immune response. Theminimum amount of Ov-ASP is the lowest amount which potentiates aspecific immune response in the subject mammal. The maximum amount ofOv-ASP is the highest amount which does not cause undesirable orintolerable side effects in the subject mammal.

The potentiation of a humoral response can be determined by measuringthe production of specific antibodies against the antigen. For example,aliquots of serum from a subject mammal can be taken and antibody titerscan be assayed during the course of an immunization program. Similarly,the presence of T cells, their effector mechanisms and/or their cytokineproducts can be monitored. For example, the potentiation of the Th1response can be determined by measuring the level of IFN-γ cytokines.The potentiation of the Th2 response can be determined by measuring thelevels of IL-4 and IL-5 cytokines. In addition, the clinical conditionsof the subject mammal can be monitored for the desired effect, e.g., aninhibition or prevention or treatment of a disease process.

The magnitude of a specific immune response is manifested by theantibody titer produced, the duration of the response, and/or thequality of the response. The magnitude of the immune response elicitedby an antigenic moiety administered along with Ov-ASP is greater thanthe immune response elicited by the antigenic moiety administered alone.

Preventing a disease means that either the mammal does not acquire thesymptoms of a disease, or that the mammal acquires fewer or less severesymptoms than the mammal would otherwise acquire without the vaccinecomposition. Treating a disease means that the mammal ceases to sufferfrom the symptoms of the disease, or that the severity of the sufferingis at least partially alleviated.

Examples of infectious diseases for which the methods of the inventionare effective are those diseases caused by the microorganisms listedabove. Examples of diseases for which the methods of the invention areparticularly effective include SARS and HIV.

Examples of tumor-associated diseases which the methods of the inventioncan treat and/or prevent include cancers of oral cavity and pharynx(i.e., tongue, mouth, pharynx), digestive system (e.g., esophagus,stomach, small intestine, colon, rectum, anus, liver, gallbladder,pancreas), respiratory system (e.g., larynx, lung), bones, joints, softtissues, skin, melanoma, breast, reproductive organs (e.g., cervix,endometirum, ovary, prostate, testis), urinary system (e.g., urinarybladder, kidney, ureter, and other urinary organs), eye, brain,endocrine system (e.g., thyroid and other endocrine), lymphoma (e.g.,Hodgkin's disease, non-Hodgkin's lymphoma), multiple myeloma, leukemia(e.g., acute lymphocytic leukemia, chronic lymphocytic leukemia, acutemyeloid leukemia, chronic myeloid leukemia).

The vaccination or administration parameters for a particular antigen,e.g., the amount of Ov-ASP to be added to particular antigen, the dosingschedule, etc., can be determined by routine experimentation. Forexample, the total amount of a vaccine composition or immunogeniccomposition and the relative amounts of an antigen and Ov-ASP within acomposition can be determined by testing the compositions in mammaliansubjects. A mammalian subject can initially be given a low dose of thecomposition and then the dose and/or the relative amounts of the proteinadjuvant and antigen can be varied while monitoring the immune response.

If inadequate vaccination or immune response is achieved then thevaccination or administration parameters can be modified in a fashionexpected to potentiate the immune response, e.g., by increasing theamount of antigen and/or of Ov-ASP, by complexing the antigen with acarrier, by conjugating the antigen to an immunogenic protein, or byvarying the route of administration, as is known in the art.

Methods of Stimulating a Cellular Response with Cytokine Secretion

Another embodiment of the present invention includes a method ofstimulating a cellular response with cytokine secretion in a mammal inneed thereof. The method comprises administering to the mammal aneffective amount of Ov-ASP, or at least one subunit of Ov-ASP. Ov-ASPcan be administered in the form of a pharmaceutical composition.

The cellular response is stimulated in mammals whether or not they havebeen previously exposed to the parasite from which Ov-ASP is derived.This unexpected discovery demonstrates that the stimulation of acellular response by Ov-ASP is not an adaptive immune response (i.e., isnot manifested by immunological memory). Instead, the cellular responseis due to stimulation of the innate immune response.

An effective amount of Ov-ASP is any amount which upregulates theinfection-clearing aspects of the innate immune response. Theupregulation of the infection-clearing aspects of the innate immuneresponse includes the induction of the inflammatory response, theregulation of hematopoiesis, the control of cellular proliferation anddifferentiation, and the healing of wounds. The minimum amount of Ov-ASPis any amount which upregulates these processes. The maximum amount ofOv-ASP is an amount which does not cause excessive proinflammatoryeffects.

The administration of Ov-ASP can be effected by administering theprotein itself, or by introducing a nucleic acid encoding the protein ina manner permitting expression of the protein, as described above.

The particular amount of Ov-ASP administered depends upon the subjectmammal being treated, the route of administration, and the pathology forwhich the mammal is being treated. For example, Ov-ASP could be injectedinto a tumor or applied to the site of a herpes virus infection tostimulate cytotoxic cellular responses that would diminish the tumor orhelp clear the virus infection.

Ov-ASP stimulates Th1, Th2, and regulatory Th cells via IL-10 cellularresponses. However, the protein predominantly stimulates Th1 responses.

Th1 responses are especially effective for inducing antitumor responses.For example, Th2 polarized responses are elicited in patients withactive cancer. Successful therapy in some cancer patients has found tobe accompanied by a shift from a Th2 polarization to a Th1 polarization.Additionally, since allergens induce Th2 responses, the administrationof Ov-ASP can be used to inhibit allergic responses by biasing responsesto Th1.

The cytokines which are stimulated by Ov-ASP include interferon-gamma(IFN-γ), granulocyte-macrophage colony-stimulating factor (GM-CSF),tumor necrosis factor-alpha (TNF-α), tumor growth factor-beta (TGF-β),interleukin-10 (IL-10), or combinations thereof.

Methods of Vaccinating or Generating an Immune Response againstOnchocerciasis

Another embodiment of the present invention includes a method ofvaccinating or generating an immune response against Onchocerciasis in amammal. The method comprises administering to a mammal, in need thereof,an effective amount of Ov-ASP, or immunogenic fragments of Ov-ASP.Ov-ASP can be administered by itself or with an adjuvant.

Onchocerciasis, or River Blindness, occurs primarily as a result of ahost inflammatory response to infection with the filarial nematodeOnchocerca volvulus. Transmitted by the bites of blackflies from thefamily Simuliidae, the parasite invades the skin, subcutaneous tissues,and other tissues, producing fibrous nodules. The host inflammatoryresponse to infection with Onchocerca volvulus can manifest in chronicskin disease and eye lesions.

An effective amount of Ov-ASP is an amount that prevents or inhibitsOnchocerciasis. For this purpose, it is necessary for the protein toproduce cytophilic antibodies. Cytophilic antibodies are antibodies thatin partnership with effector cells such as neutrophils, macrophagesand/or eosinophils, for example, can significantly inhibit the growth ofand/or kill the parasite. Growth is significantly inhibited if theinhibition is sufficient to prevent or reduce the symptoms of thedisease in an infected mammal.

The administration of Ov-ASP can be effected by administering theprotein itself, or by introducing a nucleic acid encoding the protein ina manner permitting expression of the protein, as described above.

General Methods

A mammal which can benefit from the methods of the present invention canbe any mammal. Categories of mammals include humans, non-human primates,livestock, domestic mammals, laboratory mammals, etc. Some examples oflivestock include cows, pigs, horses, goats, cattle, etc. Some examplesof domestic mammals include dogs, cats, etc. Some examples of laboratorymammals include rats, mice, rabbits, guinea pigs, etc.

A mammal in need of the methods of this invention include mammals inwhich the prevention or a treatment of a disease is desired. The diseasecan be an infectious disease; an allergy; a tumor-associated disease,such as cancer; and/or an autoimmune disease.

The pharmaceutical and vaccine compositions of the present invention canbe administered by any means as long as the administration results inthe desired immune response. Preferably, the compositions areadministered intramuscularly, subcutaneously, transdermally,intranasally, transmucosally, intraocularly, intraperitoneally, orallyor intravenously. Other suitable routes of administration include byinhalation, intratracheally, vaginally, rectally, and intraintestinally.

The means of administration of the compositions include, but not limitedto, needle injection, catheter infusion, biolistic injectors, particleaccelerators (i.e., “gene guns” or pneumatic “needleless” injectors--forexample, Med-E-Jet (Vahlsing, H., et al., J. Immunol. Methods 171,11-22(1994)), Pigjet (Schrijver, R., et al., Vaccine 15, 1908-1916 (1997)),Biojector (Davis, H., et al., Vaccine 12, 1503-1509 (1994)); Gramzinski,R., et al., Mol. Med. 4, 109-118 (1998)), AdvantaJet, Medijector,gelfoam sponge depots, other commercially available depot materials(e.g., hydrogels), osmotic pumps (e.g., Alza minipumps), oral orsuppositorial solid (tablet or pill) pharmaceutical formulations,topical skin creams, and decanting, use of polynucleotide coated suture(Qin et al., Life Sciences 65, 2193-2203 (1999)) or topical applicationsduring surgery.

The pharmaceutical and vaccine compositions of the present invention canbe formulated according to known methods. For example, the compositionscan comprise a suitable carrier. Suitable carriers include any of thestandard pharmaceutically acceptable carriers, such as water, phosphatebuffered saline solution, and aluminum hydroxide, latex particles,bentonite, liposomes and microparticles. Suitable carriers aredescribed, for example, in Remington's Pharmaceutical Sciences, 16thEdition, A. Osol, ed., Mack Publishing Co., Easton, Pa. (1980), andRemington's Pharmaceutical Sciences, 19th Edition, A. R. Gennaro, ed.,Mack Publishing Co., Easton, Pa. (1995). The pharmaceutical compositioncan be formulated as an emulsion, gel, solution, suspension, lyophilizedform, or any other form known in the art.

The vaccine compositions or immunogenic compositions of the presentinvention can comprise adjuvants. In the embodiment wherein Ov-ASP isused as an adjuvant, other additional adjuvants can be included.Examples of adjuvants include muramyl peptides and analogues;lymphokines, such as interferon, interleukin-1 and interleukin-6;saponins, fractions of saponins; synthesized components of saponins;pluronic polyols; trehalose dimycolate; amine containing compounds;cytokines; and lipopolysaccharide derivatives.

In addition, the vaccine and pharmaceutical composition can also containpharmaceutically acceptable additives including, for example, diluents,binders, stabilizers, and preservatives.

The vaccine and pharmaceutical compositions can also comprisetherapeutic ingredients. For example, formulations suitable forinjection or infusion include aqueous and non-aqueous sterile injectionsolutions which may optionally contain antioxidants, buffers,bacteriostats and solutes which render the formulations isotonic withthe blood of the intended recipient, and aqueous and non-aqueous sterilesuspensions which can include suspending agents and thickening agents.

The vaccine and pharmaceutical compositions can be presented inunit-dose or multi-dose containers, for example, sealed ampoules andvials, and may be stored in a freeze-dried (lyophilized) conditionrequiring only the addition of the sterile liquid carrier, for example,water for injection, immediately prior to use.

The invention will be more fully understood in the light of thefollowing examples. All literature and patent document citations areexpressly incorporated by reference.

EXAMPLES

The Examples demonstrate that rOv-ASP-1 acts as a potentimmunostimulator as well as an adjuvant. The Examples also demonstratethat rOv-ASP-1 is a potent stimulator of cytokine secretion in humans,whether or not they have been exposed to the parasite from which theprotein was cloned. The adjuvant properties of rOv-ASP-1 in mice andimmunostimulatory activity on human leukocytes have been shown to not bedue to contaminating bacterial lipopolysaccharide (LPS), also known asendotoxin.

Example 1

Human Cytokine Responses to rOv-ASP-1.

Experiments investigating immune responses to rOv-ASP-1 in humansubjects living in areas of Africa endemic for Onchocerca volvulus wereconducted. During these studies, it was noted that the recombinantprotein stimulated potent cytokine responses from control subjects whoresided in the New York metropolitan area and who were never exposed tothe parasite (FIG. 1). The recombinant protein stimulated significant(P<0.05) production of Th1-type cytokines (i.e. IFN-γ, GM-CSF andTNF-α), and a Th2/regulatory T cell cytokine (IL-10).

One concern was that residual LPS (endotoxin) derived from the bacteriaE. coli in which rOv-ASP-1 was cloned could be contributing to thecytokine stimulating effect. Even though the optimal cytokine-inducingconcentration of Ov-ASP-1 (5 μg/mL) tested negative for LPS activity inthe Limulus amebocyte lysate (LAL) assay (Sigma, St. Louis, Mo.),further action was taken to ensure that the results were not due to anyresidual LPS in the antigen preparation. The data presented in FIG. 2shows that the bioactivity of rOv-ASP-1 was not due to any possible LPScontamination since the cytokine production by human PBMC was notaffected by the presence of polymyxin B (Sigma), an inhibitor of LPSactivity.

Binding of rOv-ASP-1 to Human Peripheral Blood Mononuclear Cells.

The cells in peripheral blood mononuclear cells (PBMC) that bound therecombinant protein were identified using biotin-labeled rOv-ASP-1. Asshown in Table 1, rOv-ASP-1 bound to most B cells and monocytes(>94.5%). In addition, 14.5% of CD8+ T cells and 28.7% of NK cells boundthe protein. CD8+ T cells and NK cells are the likely sources of theIFN-γ secretion induced by rOv-ASP-1.

TABLE 1 FACS analysis of binding of FITC-labeled biotinylated rOv-ASP-1to subsets of human leukocytes in PBMC. Samples 1 and 2 were obtainedfrom separate donors and 10,000 events were counted. Values representthe % of total cells gated for a particular CD marker that also boundFITCbiotin-rOv-ASP-1. % rOv-ASP-1 Positive cells Cell population CDmarker Donor # 1 Donor # 2 Average T cells CD4 3.8 2.3 3.0 T cells CD816.7 12.4 14.5 B cells CD19 96.1 93.0 94.5 NK cells CD56 30.9 26.5 28.7monocytes CD14 98.6 97.9 98.3

Mouse Antibody and Cytokine Responses to rOv-ASP-1.

While conducting experiments designed to evaluate rOv-ASP-1 as apossible vaccine candidate against onchocerciasis in humans, BALBC/cByJmice were vaccinated with the recombinant protein alone or withadjuvants. IgG1 and IgG2a isotypes were measured which are associatedwith Th2 and Th1 helper T cell responses, respectively, in mice. Broadlyspeaking, Th2 immune responses are active against extracellularpathogens in the tissue fluids and Th1 responses are most effectiveagainst pathogens that infect cells.

Even without adjuvants, rOv-ASP-1 was able to stimulate high titers ofantibodies to itself in vaccinated mice.(Table 2). The proteinstimulated both Th2 (IgG1) and Th1 (IgG2a) antibodies, with a slight Th1dominance.

Spleen cells were collected from these mice in order to assess thecellular responses induced by rOv-ASP-1 to the protein. The spleen cellswere cultured and re-stimulated in vitro with rOv-ASP-1.Interferon-gamma (IFN-γ) was measured as a marker for a Th1 response.Interleukin-5 (IL-5) was measured to indicate Th2 activity. IL-10 wasmeasured as a Th2 and/or regulatory T cell product. The recombinantprotein stimulated high levels of IFN-γ secretion from spleen cellsobtained from mice injected with either PBS or rOv-ASP-1 (FIG. 3),implying direct induction of these cytokines in vitro. A similarnon-antigen-specific release of IL-10 also occurred. In contrast, IL-5was produced only by spleen cells from the mice previously exposed torOv-ASP-1, particularly by the group that received 2.5 μg of rOv-ASP-1,indicating antigen specificity of the IL-5 response.

TABLE 2 Reciprocal end-point titers of IgG1 and IgG2a antibodies torOv-ASP-1 in mice vaccinated with the protein in PBS or PBS alone.Titers were obtained using pooled serum samples (6 mice per group). IgG1IgG2a PBS 0 0 rOv-ASP-1 in PBS 293,000 656,000

Adjuvant Studies in Mice.

Since rOv-ASP-1 was able to stimulate high-titer antibody responses toitself without added adjuvant, the question whether the protein couldact as an adjuvant for antibody responses to unrelated proteins wasinvestigated. Chicken egg albumin, also known as ovalbumin (OVA), wasused as a model antigen that does not stimulate appreciable antibodyresponses when injected into mice without adjuvants. OVA was mixed withthe commercially prepared adjuvants, alum (Sigma) or MPL+TDM (Sigma) orwith the test adjuvant, rOv-ASP-1. Five groups of mice were injectedsubcutaneously with the commercially prepared adjuvants or, for controlpurposes, with OVA or with the adjuvants alone.

Each animal received 50 μg of OVA per immunization. OVA and rOv-ASP-1were diluted in sterile, LPS- free phosphate-buffered saline (PBS).

Mice received a booster immunization after 14 days. Ten days later,serum was collected from the mice. The amounts of IgG antibodies in theserum were quantified by ELISA.

When rOv-ASP-1 was used at 25 μg/mouse, the protein (FIG. 4, blacksquares) surpassed the commercially prepared alum and MPL+TDM adjuvantsin potency. The IgG1 anti-OVA end-point titer using rOv-ASP-1 as theadjuvant at 25 μg/mouse was 102,400. The titers obtained using MPL+TDMor alum adjuvants were 18,000 and 15,000, respectively. At the lowerconcentration of rOv-ASP-1 (2.5 μg), the antiOVA titer was 8,000. TheIgG2a titers were considerably lower than those of IgG1 and only therOv-ASP-1 at 25 μg induced an appreciable anti-OVA titer (25,600).

To exclude any possibility of residual LPS in rOv-ASP-1 contributing toits adjuvant effects, LPS was removed from the concentrated stocksolution (2.5 mg/mL) of rOvASP-1 using a Detoxi-gel™ system (PierceBiotechnology, Rockford, Ill.). The adjuvancy of working dilutions ofLPS-free and LPS-containing batches of rOvASP-1 were compared. In FIG.5, the open squares show that the LPS-free rOvASP-1 performed betterthan the same protein prepared from stock containing LPS (FIG. 5, solidcircles) in augmenting antibody responses to OVA in immunized mice. Theend-point antibody titer was not obtained, but the differences areclear, especially with the IgG2a isotype. Therefore, LPS as acontributing factor to the adjuvant properties of the recombinantOv-ASP-1 protein was ruled out.

Cellular responses to the immunizing antigen, OVA, were assessed bymeasuring cytokine secretion by spleen cells from the groups of micedepicted in FIG. 4 and these results are shown in FIG. 6.

OVA-specific IFN-γ production was seen only in mice that received therOv-ASP-1 test adjuvant at both concentrations and also the MPL+TDMadjuvant. IL-5 was induced in response to OVA only with alum as theadjuvant and IL-10 release was stimulated only using the commercialadjuvants but not the test adjuvant. The lack of IL-5 and IL-10 suggestsa predominantly Th1 bias to the rOv-ASP-1-guided antiOVA immuneresponse.

Example 2

Evaluation of the Adjuvanticity of rOv-ASP-1 for Pathogen Antigens

The rOv-ASP-1 protein was tested to determine if the protein had similaradjuvant potency for antigens derived from human pathogens, namelySARS-CoV and HIV-1.

BALBC/cByJ mice were immunized by using the same batch of LPS-negativerOv-ASP-1 as shown in Example 1, but mixed with 50 μg of SARS-CoV CP-1peptide (SC-1) or HIV-1-CD4 FLSC polypeptide (FLSC) instead of OVA. Allimmunized mice were given 2 boosts this time to optimize the response,i.e., a total of 3 injections of SC-1 or FLSC with rOv-ASP-1 as the testadjuvant or MPL+TDM as a control.

Using OVA as the control antigen, the end-point titers were about2,096,000 and 1,024,000 when r-Ov-ASP-1 and MPL+TDM were used asadjuvants, respectively. These total IgG titers were approximately 10times higher than in Example 1, suggesting that an additional boostsignificantly enhances antibody production. The adjuvanticity ofrOv-ASP-1 for the SC-1 peptide exceeded that of MPL+TDM judging byend-point IgG titers of 256,000 vs. 64,000, respectively (FIG. 7). Theanti-FLSC end-point IgG titers achieved using both adjuvants wereequivalent (approximately 1,024,000; FIG. 8).

The IgG isotype responses to the SC-1 peptide and the FLSC polypeptideare summarized in Table 1. The rOv-ASP-1 protein stimulated higher IgG1,IgG2a and IgG2b titers than MPL+TDM. IgG3 titers were equally low usingboth adjuvants. IgG1 titers to the FLSC polypeptide were considerablylower than those to the SC-1 peptide. MPL+TDM induced a higher IgG2btiter to FLSC than rOv-ASP-1, whereas IgG1 and IgG3 titers were the sameusing both adjuvants.

The most striking differences between the rOv-ASP-1 and MPL+TDM -inducedresponses were the lack of an IgG2a (Th1) response to SC-1 usingMPL+TDM, and the four-fold higher IgG2a response to FLSC adjuvanted byrOv-ASP-1 compared with MPL+TDM. In contrast to SC-1 and FLSC antigens,IgG2b and IgG3 antibodies to OVA were not detectable using eitherrOv-ASP-1 or MPL+TDM adjuvants (data not shown).

Each antigen model had a different behavior depending on the antigen,but the Th1 (IgG2a) response was always higher when rOv-ASP-1 was usedas an adjuvant. With FLSC as the immunogen, there was a switch in IgG2aand IgG2b antibodies between ASP-1 and Ribi adjuvants. ASP-1 favoredIgG2a and Ribi enhanced IgG2b. No IgE was detectable using rOv-ASP-1 asan adjuvant. IgM and IgA were not tested.

TABLE 3 Reciprocal end-point titers of mouse IgG isotypes to FLSC orCP-1 antigens formulated with either the rOv-ASP-1 test adjuvant or theMPL + TDM adjuvant. rOv-ASP-1 induced much higher IgG2a antibodies (Th1)to both antigens and biased IgG1 (Th2) to SC-1 peptide. Anti-FLSCAnti-SC-1 Adjuvants Adjuvants MPL + MPL + IgG isotypes rOv-ASP-1 TDMrOv-ASP-1 TDM IgG1 3,600 3,600 115,200 14,400 IgG2a 28,800 7,200 7,200 0IgG2b 3,200 25,600 1,067 334 IgG3 6,400 6,400 320 320

1. An immunogenic composition capable of inducing an immune response ina mammal against an antigen, said composition comprising an isolatedantigen unrelated to Onchocerca volvulus activation-associated secretedprotein (Ov-ASP), said antigen mixed with an adjuvant comprising aneffective amount of an isolated full-length Ov-ASP comprising the aminoacid sequence of SEQ ID NO:
 1. 2. The composition according to claim 1,wherein the antigen is a polyamino acid.
 3. The composition of claim 2,wherein the polyamino acid is an isolated SARS-CoV polyamino acid. 4.The composition of claim 3, wherein the SARS-CoV polyamino acid is theSARS-CoV SC-1 peptide.
 5. The composition of claim 2, wherein thepolyamino acid is an isolated HIV-1-CD4 polyamino acid.
 6. Thecomposition of claim 5, wherein the HIV-1-CD4 polyamino acid is theHIV-1-CD4-FLSC polypeptide.
 7. The composition of claim 1, wherein thecomposition is for administration to a mammal.
 8. The composition ofclaim 1, wherein the immune response induced against the antigen in saidmammal is a humoral immune response.
 9. The composition of claim 1,wherein the immune response induced against the antigen in said mammalis a cell-mediated immune response.
 10. The composition of claim 9,wherein the cell-mediated immune response is a Th1 response.
 11. Thecomposition of claim 9, wherein the cell-mediated immune response is aTh2 response.
 12. The composition of claim 9, wherein the cell-mediatedimmune response is a Th1 and a Th2 response.
 13. The composition ofclaim 1, wherein the antigen to the Ov-ASP ratio by weight is from 4:1to 1:1.
 14. The composition of claim 1, wherein the antigen to theOv-ASP ratio by weight is from 4:1 to 1:4.
 15. The method ofpotentiating a specific immune response to an antigen in a mammal inneed thereof, the method comprising administering to the mammal theimmunogenic composition of any one of claims 1-14.